THYROID FUNCTION EVALUATION IN PATIENTS WITH INCREASED OR DECREASED THYROXINE-BINDING PROTEIN

THE AMEBICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 50, No. 3 Copyright 1968 by The Williams & Wilkins Co. Printed in U.S.A. THYROID FUNCTION EVALUATION IN PATIENTS WITH INCREASED OR DECREASED THYROXINE-BINDING PROTEIN JERALD M. ROSENBATJM, M.D., ARTHUR F. KRIEG, M.D., JOHN B. HENRY, M.D., JAMES M. MOZLEY, PH.D., AND JOHN G. McAFEE, M.D. Departments of Pathology and Radiology, State University of New York, Upstate Medical Center, Syracuse, New York 13210 THE SERUM PROTEIN-BOUND IODINE AND RELATED METHODS Although the serum protein-bound iodine (PBI) is perhaps the most commonly used "thyroid function test," two groups of nonthyroidal factors may affect it: iodine contamination and variations in thyroxine (T 4 )- binding protein (TBP). Effects of these variables have been recently reviewed 2 and are outlined in Table 1. Contamination due to exogenous inorganic iodides may be removed by performing the PBI after butanol extraction of serum (butanol-extractable iodine (BEI)) or column chromatography (T 4 by column). The latter technic also removes contamination due to some but not all radiographic contrast media. In the future, gas chromatography may provide a definitive answer to the contamination problem. 6 In addition to iodine contamination, alterations in TBP influence the PBI and also the BEI and T 4 by column. The most important thyroxine-binding protein is thyroxinebinding globulin (TBG, an a 2 -globulin), although thyroxine-binding prealbumin also contributes. An increase in TBG causes an elevated PBI, and a decrease in TBG is associated with a depressed PBI. 10 An explanation for these alterations in concentration of PBI secondary to changes in TBG is based upon the equilibrium between protein-bound thyroxine and "free" thyroxine (Fig. 1). About 99.9 % of thyroxine is bound to TBG; less than 0.1% of thyroxine is free or unbound. There is evidence Received November 15, 1967. Dr. Rosenbaum's present address is Department of Pathology, The Springfield Hospital, Springfield, Mass. This research was supported in part by the Division of Research Facilities and Resources of the National Institutes of Health, through Grant FR-00353. 336 that it is the free thyroxine which crosses cell membranes and is physiologically active. 10 From the law of mass action, it is apparent that in vitro both the percentage and the absolute concentration of free thyroxine are directly related to concentration of total thyroxine and inversely related to TBG concentration. The PBI, BEI, and T 4 by column reflect protein-bound thyroxine, and to a lesser extent protein-bound triiodothyronine (T 3 ). They are not appreciably influenced by free thyroxine (Fig. 2). Normally, if TBG increases, the percentage and absolute amount of free thyroxine decrease. The decreased free thyroxine causes an increase in thyroid-stimulating hormone (TSH) secretion by the pituitary which normally provokes an increase in circulating thyroxine (and PBI) sufficient to restore the absolute amount of free thyroxine to normal, although the percentage may remain decreased. Thus, the patient with an elevated TBG is euthyroid with a high PBI and normal free thyroxine. Conversely, if TBG decreases, equilibrium is shifted to the right (Fig. 1). As a result, the percentage and the absolute amount of free thyroxine increase, causing a decrease in TSH and hence a decrease in circulating thyroxine (and PBI) sufficient to restore the absolute level of free hormone to normal (percentage may remain increased). Thus, the patient with decreased TBG is euthyroid with a low PBI but normal free thyroxine concentration. The T 3 Uptake Test Interpretation of this procedure is based on two facts: normally TBG is about 30% saturated with thyroxine (Fig. 2); and thyroxine is more tightly bound than is triiodothyronine. An aliquot of patient's serum is incubated in vitro with radioactive T 3 and

Sept. 1968 FREE THYROXINE INDEX AND FREE THYROXINE FACTOR 337 resin. Radioactive T 3 will be bound by unsaturated TBG (Fig. 2). The resin binds radioactive T 3 somewhat less firmly than does TBG. Hence, radioactivity of the resin, after appropriate incubation and washing, is approximately inversely related to the concentration of unsaturated TBG. Assuming a normal level of TBG, it is apparent that unsaturated TBG will be high in hypothyroidism (little T 4 present) and low in hyperthyroidism (much T 4 present). With a proportional increase in both T 4 and TBG (Fig. 3), it is apparent that there will be an absolute increase in both unsaturated TBG and PBI, decreased T 3 resin uptake, and decreased percentage of free thyroxine, but a normal absolute concentration of free thyroxine. With a proportional decrease in both T 4 and TBG (Fig. 3) there will be an absolute decrease in both unsaturated TBG and PBI, increased T 3 resin uptake, and increased percentage of free thyroxine, but a normal absolute concentration of free thyroxine. Some causes for increased or de- TABLE 1 FACTORS AFFECTING PBI AND T 3 RESIN UPTAKE Factor Iodine-containing drugs, especially radiographic contrast media TBP* with patient euthyroid: pregnancy, estrogen therapy, hereditary TBP with patient euthyroid: nephrotic syndrome, androgen therapy, hereditary T 4 therapy T 3 therapy Drugs that compete with T«for TBP binding sites: salicylates, diphenylhydantoin Effect on PBI Effect on T3 Resin Uptake Usually no change * TBP, thyroid-binding protein (includes thyroxine-binding globulin and prealbumin). TiTBP, * T 4 + TBP (Protein-bound hormone) (free thyroxine) (unsaturated TBP) (99.9%ofT 4&30%ofTBP) (0.1%oiT<) (70% of TBP) FIG. 1. Equilibrium between protein-bound thyroxine (2' 4 ) and thyroxine-binding protein {TBP). creased TBG are outlined in Table 1. In addition, some drugs occupy TBG-binding sites, giving the same effect as a decrease in TBG. In this country, the group with the greatest risk for developing thyroid disease is young women aged 15 to 40 years. In this group, pregnancy or antiovulatory drugs frequently cause increased TBG, which invalidates use of the PBI or T 3 uptake alone as measurements of thyroid function. Although methods for the measurement of free thyroxine have been developed, these procedures do not lend themselves to regular performance with ease. The most commonly used technic 9 provides an estimate of the percentage of dialyzable thyroxine, which must then be multiplied by the PBI, BEI, or T 4 by column to obtain an absolute value for free thyroxine. Two indirect ways to estimate free thyroxine have been proposed; both are based on the PBI in conjunction with the T 3 uptake test. The Free Thyroxine Index and Free Thyroxine Factor In 1965, Clark and Horn 1 proposed that the product of the PBI and T 3 uptake is proportional to the absolute concentration of free thyroxine. They reasoned that, since increased TBG normally causes decreased T 3 uptake and an elevated PBI, the product of the two measurements might remain normal as long as the changes in TBG and PBI were proportional. This product they termed the "free thyroxine index." Goolden and associates 3 and Osorio and colleagues 5 have proposed a slightly different method for estimating free thyroxine from the PBI and T 3 uptake. Their "free thyroxine factor" is derived from mathematical relationships between T 3 and unsaturated TBG on one hand and PBI and total thyroxine on the other. The purpose of this report is to compare results of the following methods for estimating free thyroxine in serum: (1) the free

338 ROSENBATJM ET AL. Vol. 50 thyroxine index of Clark and Horn, (2) the free thyroxine factor of Goolden and associates, and (3) the dialysis technic for measurement of free thyroxine. METHODS The PBI was determined by the Auto- Analyzer wet ash method (Technicon Instruments Corporation, Chauncey, N. Y.). The 131 I-T 3 uptake was performed in duplicate by a resin sponge method (Triosorb, Abbott Laboratories, Chicago, 111.). The free thyroxine was determined by a modification of the method of Sterling and Brenner 9 (Bio-Science Laboratories, Van Nuys, Calif.). The free thyroxine index was calculated as the product of PBI and T 3 resin uptake. This was also calculated with the T 3 resin uptake expressed as a percentage of a normal serum pool. 8 The free thyroxine factor was calculated according to the equation of Goolden and associates : 3 K X free T 4 = free thyroxine factor 1.53 X PBI 1090 1 _ 15.75 (T 3 + 33.5)J Blood samples were obtained from the following groups: 36 healthy volunteers, chiefly hospital employees (23 females and 13 males, IODINE COMPOUNDS AND BINDING PROTEIN IN SERUM (6 (5 - * T 14-13 - 12 II o m i- S Or UJ 10 8 - O O 6-5 - lodotyrosines (approx. 0.3 fig/iooml.) Bound T3 fopprox. 0.3 fig/ 100 ml.) 4 -!<n ui <-> >. a. <o x Bound TQ (approx. 5.0 fig/iooml.) 2- Free T 4 fopprox. 2.0 m fig / 100 ml.) Free T3 (approx. 2.0 mfig/ 100 ml.) Serum Inorganic I fopprox. O.I-0.6 fig/iooml.) FIG. 2. Relationships between protein-bound iodine, butanol extractable iodine, thyroxine-binding globulin, and unsaturated thyroxine-binding globulin.

Sept. 1968 FREE THYROXINE INDEX AND FREE THYROXINE FACTOR 339 i\ y ' V / y NORMAL TBG HIGH TBG 3 a 3 ---""'"". A 5 C, f - ~~~~~ PBI a. 1 -LOW PBI, * ' ' NORMAL HIGH w \< -Free T4 normal in all, because ratio PBI'TBG remains normal. FIG. 3. Proportional increase and decrease in TBG and PBI with constant concentration of free thyroxine. 20 to 45 years of age); 72 unselected patients seen in our laboratory during a 3-month period for whom a free thyroxine test was requested; seven women with normal pregnancies (duration 12 to 37 weeks); 10 women who had been taking oral contraceptives for at least 4 months; and 11 clinically euthyroid "sick" patients with various conditions including cirrhosis, renal insufficiency, metastatic carcinoma, and chronic respiratory disease. RESULTS Figure 4 is a scattergram of free thyroxine versus free thyroxine index for the group of 72 unselected patients. The correlation coefficient is 0.87. When T 3 uptake was expressed as a percentage of a pooled serum control, a similar relationship was found and the correlation coefficient was O.SS. A few points are somewhat distant from the line of best fit; the reasons for these deviations were not investigated. Figure 5 is a scattergram of free thyroxine versus free thyroxine factor for the same group of 72 unselected patients. The correlation coefficient is 0.91; again a few deviant points were observed. Table 2 shows the mean ± 1 S.D. for PBI, T 3 resin uptake, free thyroxine concentration, free thyroxine index, and free thyroxine factor in the three other groups: normal pregnancy, antiovulatory therapy, and clinically euthyroid sick patients. This information is also presented in Figure 6. Generally, women with normal pregnancy or taking antiovulatory drugs had elevated PBI's; those in the euthyroid sick group often had elevated PBI's but were more variable. Specifically, the PBI was elevated in 100% of the pregnant group, 70% of those taking antiovulatory medication, and in 46 % of the euthyroid sick group. Generally, women with normal pregnancy or talcing antiovulatory drugs had low T 3 resin uptakes; the euthyroid sick group

340 ROSENBAUM ET AL. Vol. 50 FREE T 4 INDEX (PBI x T 3 ) 1000 900 800-700 - 600 500 r = 0.87 y = 74.12 x +98.87 S.E. = 61.93 n= 72 400 300-200 100 2K_ 2 3 4 5 6 7 FREE T 4 (m/ig/iooml) FIG. 4. Correlation of free thyroxine index and free thyroxine concentration in 72 unselected patients. The line of best fit ± 2 S.E. of the estimate is shown. tended to have high T 3 resin uptakes, but showed considerable variability. Specifically, the T 3 resin uptake was decreased in 100% of the pregnant group and in SO % of those taking antiovulatory medication, whereas it was normal or elevated in 100% of euthyroid sick patients. Similar patterns were noted when the T3 resin uptake was expressed as a percentage of a normal serum pool. In contrast to the PBI and T 3 resin uptake, free thyroxine (dialysis technic) was normal in women with normal pregnancy or receiving antiovulatory therapy (Fig. 6). However, seven of 11 patients (64%) in the euthyroid sick group had elevated values for free thyroxine. In the three groups of patients, the free thyroxine index and free thyroxine factor gave results which appeared similar to dialysis measurements of free thyroxine (Fig. 6). In the pregnant group the free thyroxine factor appears to be slightly lower than the free thyroxine index, as noted by Goolden and co-workers. 3 DISCUSSION Although the PBI is perhaps the most widely used measurement of thyroid function, it reflects primarily protein-bound thyroxine (99.9% of total T 4 ), rather than the physiologically active free thyroxine (0.1% of total T 4 ) (Fig. 2). Normally, if there is an increase or decrease in thyroxinebinding globulin, a similar, proportional change in PBI occurs, in order to maintain a normal concentration of free thyroxine (Fig. 3). In euthyroid women who are pregnant or taking antiovulatory drugs, increases in TBG normally occur and cause increased PBI; a "normal" PBI in these women may be associated with hypothyroidism. Measurement of free thyroxine obviates this problem but is not widely performed. In contrast to the PBI, the T 3 resin uptake shows a decrease with increased TBG and an increase with decreased TBG (Figs. 2 and 3). Therefore, the product of PBI and T 3 resin uptake (free thyroxine index) has been used

Sept. 1968 FREE THYROXINE INDEX AND FREE THYROXINE FACTOR 341 as an estimate of free thyroxine. 1 Alternatively, a free thyroxine factor may be calculated from the PBI and T 3 resin uptake. 3 In the present series, the free thyroxine (measured by dialysis technic), free thyroxine index, and free thyroxine factor gave similar results in clinically euthyroid women who were pregnant or taking antiovulatory drugs. The PBI and T 3 resin uptake alone, however, would have been misleading in these patients. In euthyroid sick patients, our findings tend to confirm previous studies 4 ' 7 ' 9 which demonstrated frequent elevations of free thyroxine. The free thyroxine index and the free thyroxine factor were also increased in most of these patients. These patients suffered from a variety of diseases; the one obvious common denominator was that they were all very ill but clinically euthyroid. Although decreased thyroxine-binding prealbumin has been demonstrated 7 in various 7 r FREE T 4 FACTOR 6-5 - r= 0.91 y = 0.51 S.E. = 0.34 n= 72 x -0.11 X, j 4 - - 3 - *./ X 2 - x > X X V *Yx i i X 1 1 1 1 1 1 FREE T 4 (m M g/iooml) FIG. 5. Correlation of free thyroxine factor and free thyroxine concentration in 72 unselected patients. The line of best fit ± 2 S.E. of the estimate is shown. TABLE 2 MEAN ± 1 S.D. OF THYROID PARAMETERS AS DETERMINED IN FOUR CATEGORIES OP INDIVIDUALS Group (No. of Subjects) PBI T3 Resin Uptake Free Thyroxine Free Thyroxine Index Free Thyroxine Factor Normal controls (36) Normal pregnancy (7) Antiovulatory therapy (10) Euthyroid "sick" (11) HI./100 ml. 7.0 ± 1.1 10.5 ± 1.1 9.8 ± 1.4 9.7 ± 4.7 % 32.1 ± 3.2 19.1 ± 2.6 24.6 ± 2.1 40.0 ± 6.9 mug./100 ml. 1.6 ± 0.3 1.7 ± 0.3 1.6 ± 0.2 3.0 ± 2.3 223.4 ± 35.6 200.0 ± 32.9 239.9 ± 38.8 398.4 ± 236.8 0.728 ± 0.157 0.49S ± 0.098 0.650 ± 0.102 1.873 ± 1.407

342 ROSENBAUM ET AL. Vol. 50 16 6 4 PBI (/ig/iooml) A B 14 12 10 8-1 c * * Resin Uptake % A B V :! c t :- 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Free T 4 (m/ig/iooml), 1 1 1 1, > B c t A t I 1 Free T 4 1 ndex B._,_ _ T c t«t 1. 48- - 44- - 40-36- 32- - 28-24- 20-640- 570-500-. 1 430-360- : «,: #^ 290-220- 4.5-4.0-3.5-3.0-2.5-2.0-1.5-1.0- Free T 4 Factor A B C =J $< til LEGEND: A= NORMAL PREGNANCY B = ANTIOVULATORY THERAPY C = EUTHYROID "SICK" PATIENTS ~~ =NORMAL RANGE 1? IG. 6. Results of PBI, T 3 resin uptake, free thyroxine concentration, free thyroxine index, and free thyroxine factor in A, pregnant women, B, women receiving antiovulatory therapy, and C, euthyroid "sick" patients. noiithyroidal diseases, this probably does not completely explain the frequent elevations of free thyroxine in this group. Perhaps, as Sterling and Brenner 9 have suggested, there may be rate-limiting reactions in hepatic and peripheral tissues which are important in determining the turnover of unbound thyroxine regardless of its level in the blood. Indications for Determining Free Thyroxine Index or Free Thyroxine Factor In the classical case of thyrotoxicosis or myxedema, the diagnosis is clinically obvious and measurement of PBI is confirmatory. However, in borderline cases, and in patients with an abnormal level of TBP, an estimate of free thyroxine may be helpful. The free thyroxine index and free thyroxine factor generally appear to correlate well with the free thyroxine concentration (correlation coefficients of 0.87 and 0.91) with occasional exceptions (Figs. 4 and 5). Free Thyroxine Index versus Free Thyroxine Factor The free thyroxine index assumes that the unbound TBP capacity is equal to the reciprocal of the T 3 resin uptake according to the equation y = ax, rather than the equation y = ax + o as used in calculating the free thyroxine factor. It has been suggested that this simplification may lead to significant errors in some patients with markedly abnormal TBP, and that the free thyroxine factor may be preferable. 3 In this small series, however, the extra effort involved in calculating the free thyroxine factor produced only slightly better correlation with free thyroxine as measured by dialysis. Iodine Contamination Measurements of free thyroxine index and free thyroxine factor as described are valid only when the PBI determination is valid; that is, iodide contamination must be ab-

Sept. 1968 FREE THYROXINE INDEX AN: sent. The problem of contamination by inorganic iodides may be circumvented by determining PBI after column chromatography (T 4 by column) or after butanol extraction. Gross contamination by organic iodides invariably invalidates the BEI, however, and may affect the T 4 by column as well. SUMMARY The serum protein-bound iodine and T 3 resin uptake measurements are discussed. Although clinically useful, these measurements may be misleading if there is an increase or decrease in thyroxine-binding globulin. In such cases, measurement of free thyroxine concentration may be indicated. The product of the serum protein-bound iodine and T 3 uptake (free thyroxine index) and the free thyroxine factor appear to correlate well with the free thyroxine concentration. Either the free thyroxine index or free thyroxine factor may be used to provide an estimate of free thyroxine concentration, and either may be easily calculated in laboratories performing accurate serum proteinbound iodine and T 3 uptake determinations. As with other measurements, it may be advisable for each laboratory to establish its own range of normal values. FREE THYROXINE FACTOR 343 REFERENCES 1. Clark, F., and Horn, D. B.: Assessment of thyroid function by the combined use of the serum protein-bound iodine and resin uptake of 131 I-triiodothyronine. J. Clin. Endocrinol., 35: 39-45, 1965. 2. Davis, P. J.: Factors affecting the determination of the serum protein-bound iodine. Am. J. Med., Ifi: 918-940, 1966. 3. Goolden, A. W. G., Gartside, J. M., and Sanderson, C: Thyroid status in pregnancy and in women taking oral contraceptives. Lancet, 1: 12-15, 1967. 4. Oppenheimer, J. H., Squef, R., Surks, M. I., and Hauer, II.: Binding of thyroxine by serum proteins evaluated by equilibrium dialysis and electrophoretic techniques. Alterations in non-thyroidal illness. J. Clin. Invest., Jfi: 1769-1782, 1903. 5. Osorio, C, Jackson, D. J., Gartside, J. M., and Goolden, A. W. G.: The assessment of free thyroxine in plasma. Clin. Sc, S3: 525-530, 1962. 6. Richards, A. II., and Mason, W. B.: Gas chromatographic separation of some iodine compounds of serum. Anal. Chem., 88: 1751-1752, 1966. 7. Richards, J. B., Dowling, J. T., and Ingbar, S. H.: Alterations in the plasma transport of thyroxine in sick patients and their relation to the abnormality in Graves' disease. J. Clin. Invest., 38:1035,1959. 8. Selenkow, H. A.: Thyroid hormone binding. J. A. M. A., 197: 669, 1966. 9. Sterling, K., and Brenner, M. A.: Free thyroxine in human serum: simplified measurement with the aid of magnesium precipitation. J. Clin. Invest., 1,5: 153-163, 1966. 10. Sterling, K., and Hegedus, A.: Measurement of free thyroxine concentration in human serum. J. Clin. Invest., 1,1: 1031-1040, 1962.